SU454736A3 - The method of producing peptides - Google Patents

Description

one

The invention relates to the preparation of peptides on iolimer carriers in a homogeneous phase.

A known method of producing peptides on a polymeric carrier in an organic solvent is that the N-protected amino acid as a salt is condensed by heating in an organic solvent with chloromethylated polystyrene. N-protection is removed by known methods and the necessary amino acid sequence is created by condensation of a polymer — amino acids with the corresponding, M-zodischennyh amino acids in solution. At each stage, the polymer - peptide is separated from the low molecular weight substances by precipitation. After the amino acid sequence has been built, the peptide is separated from the polymer. The application of the known method is associated with some difficulties. First, the attachment of the first amino acid to the polymer requires relatively harsh reaction conditions (heated in the presence of triethylamine). Secondly, the polymerpeptide is not completely precipitated after each stage, it is fractionated and

sediment entrainment of low molecular weight impurities. This leads to losses of the target peptide and its contamination.

The proposed method of obtaining allows

avoid these difficulties. The N-protected C-terminal amino acid of the created peptide is bound under mild conditions in solution by an ester bond to a hydroxyl-containing carrier polymer with a mol. at.

10,000-100,000. Polyethylene glycols, their esters with citric acid, copolymers of polyethylene glycol with succinic acid, etc. can be used as a carrier polymer.

the ether bond can be carried out, for example, with dicyclohexylcarbodiimide. You can also pre-enter the C-terminal amino acid in the monomer, and then to obtain a polymer carrier with attached

amino acid. N-protection with polymer-amino acids is removed, for example, by acidolysis or hydrogenation. The polymer is separated from the low molecular weight substances by dialysis, ultrafiltration, column chromatography or countercurrent distribution, rather than precipitation, as in the known process. Further stepwise extension of the peptide chain is carried out as usual, using dicyclohexylcarbodiimide, alkyl chloroformate, azides or activated esters of N-protected amino acids to form the amide bond. The reaction is carried out in solution in organic solvents. For water-soluble carrier polymers, the formation of an amide bond in an aqueous medium is possible. In this case, water-soluble carbodiimide is used as a condensing agent. After the capping step of condensation, the N-protection is cleaved in a suitable way and the polymer is separated from the isomolecular substances. Destruction of the bond between the prepared peptide and the carrier polymer is carried out using known methods, such as alkaline hydrolysis or ammonolysis. The peptide is separated from the polymer by one of the methods indicated.

Example 1. Esterification of BOC -valyl-OH with PET (mol. Century 20,000).

20 g of PEG, 4.34 g of BOC-valyl-OH and 4.12 g of DCC are dissolved in 200 ml of CH2CI2 and stirred at room temperature without moisture for 5.5 days. The reaction mixture is evaporated in vacuo to dryness, 100 ml 4 n is added. HCl / dioxane, stirred at room temperature for 30 minutes and evaporated to dryness in vacuo. The residue is dissolved irimerically in 1 liter of water. The precipitate which forms therewith is separated by filter aid. The filtrate is adjusted to pH 6 with NaOn or triethylamine and ultrafiltered through a Diaflo UM 10 ultrafilter while free valine can be detected in the filtrate. The filtrate is evaporated in vacuo to dryness, the residue is dried with a mixture of benzene and absolute ethanol (80/20) to constant weight. Exit H-valyl-PEG 19.35g. The amino acid content is 80%, or 0.08 mmol / g (amino acid analysis).

B. Synthesis of peptide. 5 g of H-valpl-PEG is dissolved in 50 ml of CH2Cl2 and 0.1 ml of triethylamine is added. After adding 0.44 g of BOCglycyl-OH (2.5 mmol) and 0.515 g of DCC (2.5 mmol), the mixture is stirred at room temperature for 2 hours, evaporated to dryness in vacuum, and 40 ml of 4N is treated at room temperature for 30 minutes. PS1 / dioxane and again evaporated to dryness in vacuo. The oily residue is dissolved in 500-700 ml of water, the precipitate is separated, the filtrate is adjusted to pH 6 with NaOH or triethylamine and ultrafiltrant is applied through a Diaflo ultrafilter UM 2. The filtrate is treated. The output of N-glycyl-valyl-PEG 4.85 g

In this way, triethylampn is added to H-glycyl-Valpl-PEG and reacted with 2.5 mm BOC-alanyl-OH and 2.5 mmol

 BOC - tert-butoxycarbonyl

PEG - polyether glycol glycol

DCC - dicyclohexylcarbodiimide

464736

DCC. After cleavage of the protecting group and ultrafiltration, 4.75 g of P-alanylglycyl-valyl-PEG are obtained. The corresponding interaction of P-alanyl-glycyl-valyl-PEG with BOC-leucyl-OP and DCC gives H-leucylalanyl-glycyl-valyl-PEG (4.5 g). Amino acid analysis of the hydrolyzed sample of this substance shows the following ratio of amino acids: valyl: glycyl: alanyl: leucpl 1.08: 1.00: 0.90: 1.04.

B. Cleavage of tetrapitide from the carrier. A solution of 4.0 g of C-leucyl-alanyl-glycyl-valylPEG in 75 m of water and 1.5 ml of 1N. KOH is stirred for 2 hours at room temperature, exactly neutralized with 1.5 ml of 1N. ЦС1 and ultrafiltered through Diaflo UO iO ultrafilter until 3.5 liters of filtrate have been collected. After evaporation in vacuo, it gives 280 mg of the crude product, which is partially freed from NaCl by digestion with absolute methanol. The methanol solution is provided with charcoal, filtered and evaporated to dryness. The residue is dissolved in water and lyophilized. 191.5 mg of crude tetrapeptide are obtained, the amino acid analysis of which shows the following ratio of amino acids: valyl: glycyl: alanyl: leucyl 1.03:: 1.00: 0.85: 1.07. The share of peptide in crude form is 35% (amino acid analysis), i.e., calculated for the first amino acid (valine), the total yield is 45%. The substance is identical to the known pattern obtained by the method of Merrifield. Example 2

A. To 4.34 g of BOC-valyl-OP and 2.8 ml of triethylamine in 80 ml of CH2Cl2 are added dropwise with stirring at -5 ° C 1.91 ml of ethyl chlorogolic acid ester in 20 ml of CH3C, after 30 min at -5 ° A cooled (-5 ° C) solution of 20 g of PEG (mol. C. 20,000) and 2.4 g (20 mmol) of dimethylaminopyridine in 100 ml of CH2Cl2 are added. The reaction mixture is stirred at -5 ° C for one hour and at room temperature for 65 hours. To lock

unreacted hydroxyl groups, stir the reaction mixture for 4 hours with 10 mmol of acetic anhydride and 10 mmol of triethylamine at room temperature and evaporate it in vacuo to dryness. The residue is treated for 30 min at room temperature with 100 ml 4 n. HCl in dioxane, evaporated to dryness in vacuo and dissolved in about 1 liter of water. After filtration and installation, RP will be filtered and processed as described.

above. Yield 19 g P-valyl-PEG. The amino acid content is 607о, or 0.06 mmol / g (amino acid analysis).

B. Purification of 12.7 g of P-valyl-PEG, synthesize the tetrapeptide described in Example 1B.

in the same way. Reagents: 1 mmol of triethylamine, 4.0 mmol of BOC-amino acids, 4.0 mmol of DCC. 11.5 g of P-leucyl-alanylglycyl-valyl-PEG are obtained with the following ratio of amino acids: valyl: glycyl: alanyl: leucpl 0.88: 0.86: 1.11: 1.00.

B. As described in Example 1B, 10 g of N-leucyl-alanyl-glycyl-valyl-PEG is hydrolyzed in 100 ml of water and 3 ml of 1N. KON. After neutralization, ultrafiltration and processing, 300 mg of crude tetrapeptide is obtained, which has the following ratio of amino acids: valyl: glycyl: alanyl: leucyl 0.96: 0.91:: 1.24: 1.00. For the peptide in the crude product is 41% (amino acid analysis), i.e., counting on the first amino acid (valyl), the total yield is 57% of the theoretical.

Froze To 5g of H-valyl-PEG (80%, 0.5 mmol of valine and 0.09 ml of triethylamine (0.6 mmol), dissolved in 600 ml of SPOC., 1.05 g of BOC-glycyl-OPF (3.5 mmol) are added and evaporated for 24 hours at room temperature. After evaporation in vacuum, the residue is stirred for 30 minutes at room temperature with 60 ml of trifluoroacetic acid / CH9Cl2 (1: 1) and evaporated to dryness in vacuum again. The residue is dissolved in approximately 700 ml of water, filtered and the filtrate adjusted to pH 6 with POMOSH.A and NaOH.

After ultrafiltration (diaflo ultrafilter UM 10) is treated as described in Example 1B. Output H-glycyl-valyl-PEG 5.88 g

Analogously to example 1B, N-glycyl-valylPEG is treated with BOC-leucyl-SNP. 5.6 g of N-leucyl-alanyl-glycyl-valylPEG are obtained with the following ratio of amino acids: valyl: glycyl: alanyl: leucyl 1.00: 0.70: 0.78: 0.71. 5.6 g of N-leucyl-alanyl-glycyl-valyl-PEG is hydrolyzed by printer 1B and the split tetrapeptide is treated, respectively. 250 mg of crude tetrapeptide is obtained with the following ratio of amino acids: valyl: gliyl: alanyl: leucyl 1.00:: 0.83: 0.93: 0.88. The share of the peptide in the bulk tetrapeptide is 32%, i.e., counting on the first amino acid (valine), the total yield is 50% of the theoretical.

Example 4

A. 10 g of PEG (mol. Cent. 20,000), 1.75 g of BOCglycyl-OH and 2.06 g of DCC are dissolved in 100 ml of CHdCl2 and stirred for 6 days at room temperature. The reaction mixture is evaporated in vacuo to dryness, 100 ml of 1.2 n are added. HCl in glacial acetic acid and stirred for 20 min at room temperature.

The solvent is distilled off in vacuo, and 100 ml of water is added to the residue. The resulting precipitate is separated by centrifugation. The clear upper layer was decanted, adjusted to pH 6 with triethylamine and ultrafiltered through UF Diaflo ultrafilter 10 until the ultra filtrate still contains glycyl-OH. The residue is evaporated in vacuo and traces of water are azeotroped twice with benzene. The product is dried in an exsiccator to constant weight. The output of H-glycyl-PEG 9.8 g, the amino acid content of 0.06 mmol / g

B. 9.8 g of N-glycyl-PEG is dissolved in 60 ml of CH2Cl2 and neutralized with 0.7 mmol M-meth, -;

morpholine. 3 mmol of BOC-leucyl-OH in 5 ml and 1 ml are dissolved in a separate vessel. 3 mmol of X-methylmopfoline is neuteralized, cooled to -20 ° C, 2.6 mmol of isobutyl chloroformate is added and stirred for 10 min at -20 °. The mixed anhydride is added at -20 ° C to the solution of H-glycyl-PEG and changed for an hour at -20 ° and 2 hours at 4 ° C. The solvent is distilled off, the residue is treated with 15 min at 50 ml of 1.2 n. HC1 is added to acetic acid and evaporated again in vacuo to dryness. The residue is dissolved in 100 ml of water, adjusted to pH 6 with water with sodium aietate solution and ultrapolated through a UF 10 Diaflo ultrafilter. The residue is evaporated to dryness in vacuum, twice azeotropically dried with benzene and dried in an exsiccator. The output of the N-leucyl-glyctlPEG 9.5 g

Similarly, other amino acids are combined as the following derivatives: Boc-alanyl, Boc-U-benzyl) -veonyl-OH, Boc-leucyl-OH. BOCK-valyl-ON, BOCKU-benzyl) -topeoyl-ON, BOCK-valyl-OH. After the last combination, 8.0 g are obtained.

N-valyl-toonyl-valyl-leucyl-threonylbenzyl

30benzyl

alantl-leucpl-glycyl-o-peg

Amino acid analysis of the hydrolyzed sample of this BeniecTBa gave the following result:

glycyl: leucyl: alanyl: threonyl j valyl 1.0: 1.89: 1.67: 1.0. Values for individual amino acids were obtained from the results of analyzes of amio acids on intermediate steps.

Example 5

A. Lime acid esterified into ester with PEG (mol. V. 6000) with an equimolar amount of DCC (passively on PEG) in methylene chloride (10% solution) for 3 days. The solvent is distilled off, the residue is dissolved in water and the resulting precipitate is distilled off. The solution is ultrafiltered (Amikon, UM-2-filter) is evaporated at E-vacuum and the residue is dried to a steady weight. Output 80% of theoretical. The dried polymer is esterified by BOC-valyl-OH and DNA (both components in 5-fold excess, counting at 0.2 mmol / g of functional groups on the polymep) in a 10% solution in СНоСЬ for 3 days at room temperature. The amino acid content in the polymer after the accepted treatment and cleavage of the BOC-group with 4 n. HC1 / dioxane:

: 0.2 tmol / g.

B, According to the DNA method, the following activated esters are obtained:

) 3-fold excess of PZG, counting on the functional iii.ie of the citric acid group BOC-leucyl-PCP BOC-glycyl-ONF, BOC-glutaminyl-ONF. Combinations are carried out as follows. 1 mmol (5 g) of the H-valyl polymer is dissolved in 50 ml of CH2Cl2 / dimethylformamide (1: 1), neutralized with 1.2 mmol of N-methyl morpholine and 3 mmol of the activated BOC-amino acid ester is added. After 5 minutes, a further 1.2 mmol of N-methyl morpholine was added to the solution and transferred 2 hours at room temperature. The solvent is distilled off in vacuo, 50 ml of 4 and are added to the residue. HC1 / dioxane and allowed to stand for 30 minutes at room temperature. After the second distillation of the solvent, the residue is dissolved in a water / ethanol mixture (3: 1) and ultrafiltered using a Diaflo UF-2 ultrafilter. Balance of silat. The output of the N-glycyl-leucyl-valyl polymer 4 g. The result of amino acid analysis of the hydrolyzed sample: valyl: leupyl: glycyl: glutaminyl: 1.0: 0.9: 1.0: 0.98. 2.0 g of N-glutaminyl-glycyl-leucyl-valyl-polymer is transferred for 4 hours in 0.2N. KOH (50 ml) at room temperature, neutralized with 2N. HC1 and passed through a column (2.5 x 100 cm) with Sephadex G-25. Crude ipeptide n-glutaminyl - glycyl - leucpl-valylON is obtained in a yield of about 50%. The amino acid analysis gives the following values: valyl: leucyl: glycyl: glutaminyl 1.0: 0.88: 1.05: 0.98. Example 6. A. PEG (mol. C. 4000) is esterified with a 10-fold excess of BOC-glypyl-OH and DCC in a 10% solution of CH2Cl2 for 5 days at room temperature. The protective group is then cleaved, the solvent is distilled off, the residue is dissolved in water and the resulting precipitate is separated by centrifugation. The solution is ultrafiltered with a Diaflo UM-2 ultrafilter, the solvent is distilled off and the residue is dried. The amino acid content is found by amino acid analysis as 100%. Maleic acid is reacted with n-NITROPHENOL and DCC to give maleic acid DIONPH ester. Yield about 80% of theoretical, melting point 185-190 ° C. Hydrogen iodide is added to the double bond of the maleic ester thus obtained. The resulting monoiodide succinic acid di-p-nitrophenyl ester is reacted with BOC-valyl-OH Ag salt, to obtain BOC-valyl-O-succinic acid di-UNP ester. Equimolar amounts of PEG-glycyl-N (neutralized with N-methyl-morpholine) and BOC-valyl-O-succinic acid di-ONT ester are dissolved in CHaCl2 dimethylformampds (I: 1; 10% solution) and stirred at room temperature 24 hours. After that, the solvent is distilled off, the residue is dissolved in water and ultrafiltered (Diaflo ultra), -. | 11: it: lorpheni, 1

ONF - -nitrophenyl filter UM-10) until the tolpdin test in the ultrafiltrate becomes negative. The filtrate is evaporated in vacuo and the residue is substantial. The amino group H-glycyl-PEG is then reacted with 3-sulfopropionic anhydride. From the resulting product, the BOC group is split off with 4 n. HC1 / dioxane. The resulting polymer gmset mol. at. Higher than 20,000 (according to withholding). B. 2 g of the obtained polymer and 6 mmol of BOC-leucyl-PCP are transferred to 20 ml of CH2Cl2 / dimethylformamide (1: 1) for 3 hours at room temperature (after 5 min, 2 mmol of N-methylmorpholine is added to the reaction mixture). After that, the solvent is distilled off in vacuum, the residue is treated with 20 min. 40 ml of 4 n. HC1 / dioxane and the solvent is again distilled off. The residue is dissolved in 100 ml of water and ultrafiltered (YM-10). Water is evaporated and the residue is present. Output 1.9 g. Analysis: Valyl: leucyl 1.0::: 0.95. B. 1.9 g of the dipeptide polymer is stirred for 3 hours at room temperature in 0.2 n. water KOH, neutralized 2 n. HC1 and ultrafiltered through a UM-10 filter. The filtrate is evaporated in vacuo. Crude yield 350 mg. The peptide content is 70%. Amino acid analysis: valyl: leucyl 1.0: 0.97. Example 7. First, esterified with BOCglycyl-OH with PEG (mol. C. 200) according to example 6A. Removal of excess components is carried out by ultrafiltration with a Diaflo UM-2 filter or chromatography on Sephadex. The resulting product is condensed analogously to example 6 with a di-ONP-maleic ester. Hydrogen iodide is added to the condensation product and the iodinated product is reacted with a BOC-alanyl-OH Ag salt. Dioxane is used as a solvent instead of ether. After esterification, the solvent is distilled off, the residue is dissolved in water and ultrafiltered (UM-10 filter). After residue residue, 95% of the esterified polymer is obtained. After removal, the protective group is combined with BOC-lainil-PCP. Output D111: epidepolymer 1,95 g (93% of theoretical). Amino acid analysis: alanyl: leucyl 1.0: 1.01. Cleavage of the peptide from the polymeric carrier yields 300 mg of the crude product. The yield according to amino acid analysis is 80% of the theoretical: alanyl: leuc1 l 1.0: 1.0. The peptide is homogeneous in thin layer chromatography in an ice / acetic acid (butanol) water system (3: 1: 1). Example 8. A. Yu g of PEG (mol. D. 20,000), 1.16 g of BOCIS-leucyl-OH, and 1.03 g of DCC are dissolved in 9,100 ml of CH2Cl2 and stirred for 6 days at room temperature without access of moisture. The reaction mixture is evaporated to dryness in vacuo, 50 ml 4 are added and. HCl / dioxane p is stirred for 30 minutes at room temperature. After distilling off the solvent, the residue is dissolved in 100 ml of water and the precipitated precipitate is separated by centrifugation. The separated solution is adjusted to pH 6 with the aid of p 2n. NaOH and ultrafiltered through diaflo ultrafilter UM-10. The filtrate is evaporated to dryness in vacuo and dried. Output H-isoleucyl-PEG 9.8 g. The content of isoleucil, according to amino acid analysis, is 0.07 mmol / g (70% of theoretical). B. The resulting product is combined in turn with the BOC derivatives of alanine, valine and phenylalanine following the scheme. The 10% aqueous polymer solution is adjusted to pH 3.9 with 4N. NaOH or 5 n. H2SO4 and cooled to 4 ° C. In a separate pump, the solution of BOC-ampno-acid (5-fold excess compared to the amino component) is dissolved in 2 to 3 ml of dimethylformamide and added to the aqueous polymer solution, maintaining the pH 3.9 constant. At the same pH and temperature, an equimolar BOC amino acid quantity of d-toluenesulfonate N-cyclohexyl-C- p- (N-methyl-morpholino) -ethyl-carbodiimide-p-toluenesulfonate is added with stirring, maintaining pH 3.9 with 5 n. H2SO4. After about 15 minutes, when the pH no longer changes, the pH is adjusted to 7.0 with 4N. NaOH and stirred for 30 minutes at room temperature. The pH is then again adjusted to 3.9 and an equimolar amount of condensing agent is added. The solution is left for 3 to 4 hours at pH 7.0 and at room temperature and ultrafiltered. By evaporation in vacuo, a tetrapeptide polymer is obtained. in Tetrapitp-polymer (9 g) is treated 24 hours 2 p. KOH in a mixture of water / dioxane (9 g in 50 ml) is neutralized and subjected to gel filtration through a column (2.5 x 80 cm) with Sephadex G-15 in an aqueous solution. Water is distilled off (at 40 ° C). The residue is dissolved in methanol, the solvent is distilled off and the peptide is dried. The output of raw H-alapil-Valpl-fenplizoletspl-IT: about 300 mg (not quite free from salt). Amino acid analysis gives a yield of approximately 0.5 mmol (75% of the theoretical value, considering the content of amino acids in the polymer). The result of the analysis after chromatography on a column: isoleucyl: phenyl: valyl: alaNil 1, 0: 0,99: 0,90: 0,97. Example 9. A. 20 g of PEG (mol., 20,000), 5.0 g of C-wolyl-OH and 4.2 g of DCC are dissolved in 200 ml of CH 2 C 12 and stirred for 10 days at room temperature without access to moisture. 1.2 ml of le) C - benzyloxycarbonyl 454 DANO acetic acid was added to the reaction mixture, stirred for another 20 hours and evaporated to dryness in vacuo. The residue is dissolved in 300 ml of MeOH, 5 ml of 4 N are added. HC1 / MeOH and hydrogenate above Pd / C npjt at room temperature and normal pressure while hydrogen is absorbed. The catalyst is filtered off, the filtrate is evaporated to dryness in vacuo, the residue is dissolved in approximately 1 liter of water and the precipitate formed is separated. The filtrate is filtered through a Diaflo UM-40 ultrafilter and is processed further as described in Example 1A. The output of 18.8 g of N-Vall-PEG-HC1. The amino acid content is 88%, or 0.088 mmol / g (amyio-slot analysis). B. 10 g of H-valyl-PEG-HC1 is dissolved in 50 ml of CH2Cl2, cooled to -20 ° and the solution adjusted to pH 8-9 by adding 0.11 ml of N-methylmorpholine. 1.05 g of C-glycyl-OH is dissolved in 10 ml of tetrastrofrofuran, cooled to -20 °, 0.55 ml of N-methylmorpholine and 0.65 ml of isobutyl ether of chloroformic acid are added and stirred 5 min at -20 ° . This mixture is added to the NchwalylPEG solution which has been cooled to -20 ° and stirred for 30 minutes below -10 °, 30 minutes below 0 ° and 3 hours at room temperature. The reaction mixture is evaporated to dryness in vacuo, the residue is dissolved in 200 ml of MeOH, 1.25 ml of 4N is added. HC1 / MeOH and Pd / C are hydrogenated at room temperature and normal pressure until the absorption of hydrogen is complete. The catalyst is filtered off, the filtrate is evaporated in vacuo to dryness, the residue is dissolved in about 1 l of water, filtered and ultrafiltered through a Diaflo UF-10 ultrafilter. The ultrafiltration filtrate is evaporated to dryness in a vacuum, dried with benzene / absolute ethanol (80/20) and overnight in an exsiccator to constant weight. Yield of H-glycyl-valyl-PEG-HC1 8.0 g. In a similar manner, 4.0 g of H-glycyl-valylPEG-HC1 is adjusted to pH 8-9 with 0.05 ml of N-methylmorpholine and iodized to react with the mixed anhydride made from 500 mg of C-alanyl-OH, 0.22 ml of N-methylmorpholine and 0.26 ml of isobutyl chloroformate in 5 ml of tetrahydrofuran. After cleavage of the protecting group and ultrafiltration, 3.8 g of H-alanyl-gl1-SCHIL-valyl-PEG-HC1 are obtained. The corresponding interaction of this product with C-leucyl-OH. N-methylmorpholine and isobutyl chloroformate ester are obtained after cleavage of the protective group and ultrafiltrate 3.5 g of N-leucyl-alanylglidyl-valyl-PEG HC1. The amino acid analysis of the hydrolyzed sample of this substance shows the following ratio of amino acids: valyl: glycyl:: alanyl: leucpl 1.00: 0.89: 0.92: O 94. B. 3.4 g H - leucyl - alanyl - glycyl - valylPEG- HC1 is hydrolyzed analogously to example B in 62 ml of water using 1.25 ml Jn,